Accumulation of p27 KIP1 is associated with BMP2-induced growth arrest and neuronal differentiation of human neuroblastoma-derived cell lines

Bone morphogenetic proteins (BMPs) play an essential role in cell fate determination. In this study, we found that BMP2 treatment resulted in growth arrest and differentiation in human neuroblastoma-derived cell lines, SH-SY5Y and RTBM1. Within 30min of BMP2 exposure, phosphorylation of Smad1/5 was observed in these cell lines. In RTBM1 cells, BMP2-induced differentiation was accompanied by a significant decrease in the expression level of DAN, an antagonist of BMP in frog embryos. Immunoblot analysis revealed that BMP2 treatment caused a down-regulation of p53 family members and hence of cyclin-dependent kinase inhibitor p21(WAF1). We found a significant accumulation of p27(KIP1) in response to BMP2, whereas the expression level of Skp2, which is required for ubiquitin-dependent p27(KIP1) degradation, was decreased during this differentiation process. Our results suggest that p27(KIP1) contributes to the BMP-induced growth arrest and neuronal differentiation of neuroblastoma, and BMP treatment might provide a new therapeutic strategy.     

Cytoskeleton changes following differentiation of N1E-115 neuroblastoma cell line

Summary. No systematic approach to detect expression of differentiation-related elements was published so far. The undifferentiated N1E-115 neuroblastoma cell line was switched into a neuronal phenotype by DMSO treatment and used for proteomic experiments. We used two-dimensional gel electrophoresis followed by unambiguous mass spectrometrical identification of proteins to generate a map of cytoskeleton proteins (CPs), i.e., to search for differentiation-related structures. Alpha-actin, actin-like protein 6A, gamma-tubulin complex component 2, tubulin alpha 3/alpha 7, CLIP associating protein 2, B4 integrin interactor homolog were detectable in the undifferentiated cell line exclusively and neuron-specific CPs drebrin and presynaptic density protein 95, actin-related protein 2/3, alpha and beta-centractin, PDZ-domain actin binding protein, actinin alpha 1, profilin II, ezrin, coactosin-like protein, transgelin 2, myosin light polypeptide 6, tubulin alpha 2, 6 and 7, beta tubulin (94% similar with tubulin beta-2), tubulin beta 3, tubulin tyrosine ligase-like protein 1, lamin B1 and keratin 20 were observed in the differentiated cell line only. We herein identified differentiation-related expressional patterns thus providing new evidence for the role of CPs in the process of neuronal differentiation.     

Regulation of p27 in the process of neuroblastoma N2A differentiation

Neuronal differentiation implies morphological and biochemical changes to generate a specialized neuron. N2A neuroblastoma cells can be promoted to undergo differentiation associated to neurites outgrowth, a process linked to the arrest of cell division. Using N2A cells as a model, we investigated the detailed molecular aspects on the involvement of p27 in dibutyryl cAMP-induced neuronal differentiation. In the undifferentiated N2A phenotype, an unusually high level of accumulated p27 protein mass was evidenced. Data suggest that in proliferating cells, p27 could be sequestered by direct interaction with cyclin D1, thus preventing its inhibitory action on cell cycle Cdks. Studies also indicate that p27 is functionally active and that its loss of action on Cdks in proliferating cells is due to its strong association with cyclin D1. Therefore, when cell differentiation is triggered, the action of p27 on Cdks seems to depend on both p27 and cyclin D1 degradation during the early steps of differentiation followed by late events of re-synthesis of active p27. In this context, an overexpression of p27 after N2A transfection with a mouse p27 clone induces the outgrowth of neurites associated with a decrease in cyclin D1 expression. On the other hand, treatment of N2A undifferentiated cells with c-myc antisense oligonucleotides led to a decrease in p27 and cyclin D1 levels, similar events as those in early stages of cell differentiation. Studies suggest that blockage in c-myc expression triggers early events in neuronal differentiation. These studies are of the utmost importance to elucidate regulatory mechanisms of molecules that play a critical role in the transition from a proliferating phenotype to differentiated cells.     

Inhibition of mir-21, which is up-regulated during MYCN knockdown-mediated differentiation, does not prevent differentiation of neuroblastoma cells

Background

Neuroblastoma is a malignant childhood tumour arising from precursor cells of the sympathetic nervous system. Genomic amplification of the MYCN oncogene is associated with dismal prognosis. For this group of high-risk tumours, the induction of tumour cell differentiation is part of current treatment protocols. MicroRNAs (miRNAs) are small non-coding RNA molecules that effectively reduce the translation of target mRNAs. MiRNAs play an important role in cell proliferation, apoptosis, differentiation and cancer. In this study, we investigated the role of N-myc on miRNA expression in MYCN-amplified neuroblastoma. We performed a miRNA profiling study on SK-N-BE (2) cells, and determined differentially expressed miRNAs during differentiation initiated by MYCN knockdown, using anti-MYCN short-hairpin RNA (shRNA) technology.

Results

Microarray analyses revealed 23 miRNAs differentially expressed during the MYCN knockdown-mediated neuronal differentiation of MNA neuroblastoma cells. The expression changes were bidirectional, with 11 and 12 miRNAs being up- and down-regulated, respectively. Among the down-regulated miRNAs, we found several members of the mir-17 family of miRNAs. Mir-21, an established oncomir in a variety of cancer types, became strongly up-regulated upon MYCN knockdown and the subsequent differentiation.Neither overexpression of mir-21 in the high-MYCN neuroblastoma cells, nor repression of increased mir-21 levels during MYCN knockdown-mediated differentiation had any significant effects on cell differentiation or proliferation.

Conclusions

We describe a subset of miRNAs that were altered during the N-myc deprived differentiation of MYCN-amplified neuroblastoma cells. In this context, N-myc acts as both an activator and suppressor of miRNA expression. Mir-21 was up-regulated during cell differentiation, but inhibition of mir-21 did not prevent this process. We were unable to establish a role for this miRNA during differentiation and proliferation of the two neuroblastoma cell lines used in this study.     

Intracellular distribution of functional M(1) -muscarinic acetylcholine receptors in N1E-115 neuroblastoma cells

Signaling by muscarinic agonists is thought to result from the activation of cell surface acetylcholine receptors (mAChRs) that transmit extracellular signals to intracellular systems. In N1E-115 neuroblastoma cells, we detected both plasma membrane and intracellular M(1) -mAChRs using both biochemical and pharmacological methods. In intact cells, both plasma membrane and intracellular M(1) -mAChRs were detected by the hydrophobic ligand probe, 1-quinuclidinyl-[phenyl-4-(3) H]-benzilate ([(3) H]-QNB) whereas the hydrophilic probe, 1-[N-methyl-(3) H] scopolamine ([(3) H]-NMS), detected only cell surface receptors. These probes detected comparable numbers of receptors in isolated membrane preparations. Immunohistochemical studies with M(1) -mAChR antibody also detected both cell-surface and intracellular M(1) -mAChRs. Carbachol-stimulated phosphatidylinositol hydrolysis and Ca(2+) mobilization were completely inhibited by a cell-impermeable M(1) antagonist, muscarinic toxin -7 and the G(q/11) inhibitor YM-254890. However, carbachol-stimulated extracellular-regulated kinase 1/2 activation was unaffected by muscarinic toxin-7, but was blocked by the cell-permeable antagonist, pirenzepine. extracellular regulated kinase 1/2 phosphorylation was resistant to blockade of G(q/11) (YM-254890) and protein kinase C (bisindolylmaleimide I). Our data suggest that the geographically distinct M(1) -mAChRs (cell surface versus intracellular) can signal via unique signaling pathways that are differentially sensitive to cell-impermeable versus cell-permeable antagonists. Our data are of potential physiological relevance to signaling that affects both cognitive and neurodegenerative processes.     

Up-regulation of Jun/AP-1 during differentiation of N1E-115 neuroblastoma cells.

Neuroblastoma cell lines isolated from neuroblastoma tumors can be induced to differentiate into neuronal cell types by treatment with chemical agents, such as dimethyl sulfoxide and retinoic acid. The molecular mechanisms underlying this differentiation process, however, are completely obscure. In this paper, we show that neuronal differentiation of mouse N1E-115 neuroblastoma cells by dimethyl sulfoxide is accompanied by a prolonged rise in c-jun, junB, and junD expression and AP-1 activity. Multiple sequence elements in the Jun promoters are involved in this process. Furthermore, we show that c-jun and junD, but not junB, are expressed at high levels in the neuronal cell types obtained after dimethyl sulfoxide treatment. These results suggest an important role for c-jun and junD in neuronal differentiation of N1E-115 cells.     

Quantitative proteomics reveals EVA1A‐related proteins involved in neuronal differentiation

EVA1A is an autophagy‐related protein, which plays an important role in embryonic neurogenesis. In this study, we found that loss of EVA1A could decrease neural differentiation in the brain of adult Eva1a ^?/? mice. To determine the mechanism underlying this phenotype, we performed label‐free quantitative proteomics and bioinformatics analysis using the brains of Eva1a ^?/? and wild‐type mice. We identified 11 proteins that were up‐regulated and 17 that were down‐regulated in the brains of the knockout mice compared to the wild‐type counterparts. Bioinformatics analysis indicated that biological processes, including ATP synthesis, oxidative phosphorylation, and the TCA cycle, are involved in the EVA1A regulatory network. In addition, gene set enrichment analysis showed that neurodegenerative diseases, such as Alzheimer's disease and Huntington's disease, were strongly associated with Eva1a knockout. Western blot experiments showed changes in the expression of nicotinamide nucleotide transhydrogenase, an important mitochondrial enzyme involved in the TCA cycle, in the brains of Eva1a knockout mice. Our study provides valuable information on the molecular functions and regulatory network of the Eva1a gene, as well as new perspectives on the relationship between autography‐related proteins and neural differentiation.     

Development of resting membrane potentials in differentiating murine neuroblastoma cells (N1E-115) evaluated by flow cytometry

With the aid of a voltage-sensitive oxonol dye, flow cytometry was used to measure relative changes in resting membrane potential (V_m) and forward angle light scatter (FALS) profiles of a differentiating/differentiated murine neuroblastoma cell line (N1E-115). Electrophysiological differentiation was characterized by V_m establishment. The (V_m)-time profile was found to be seed cell concentration-dependent for cell densities of less than 2 × 10⁴ cells/cm². At higher initial cell densities, under differentiating culture conditions, V_m development commenced on day 2 and reached a steady-state on day 12. The relative distribution of differentiated cells between low and high FALS has been proposed as a potential culture electrophysiological differentiation state index. These experiments offer a general methodology to characterize cultured excitable cells of nervous system origin, with respect to electrophysiological differentiation. This information is valuable in studies employing neuroblastoma cells as in vitro screening models for safety/hazard evaluation and/or risk assessment of therapeutical and industrial chemicals under development. This revised version was published online in July 2006 with corrections to the Cover Date.     

Integrin-linked kinase controls neurite outgrowth in N1E-115 neuroblastoma cells

Mouse N1E-115 cells grown on a laminin matrix exhibit neurite outgrowth in response to serum deprivation. Treatment of cells with an antibody against beta(1) integrin inhibits neurite outgrowth. Thus, beta(1) integrin is involved in the neuritogenesis of N1E-115 cells on a laminin matrix. Integrin-linked kinase (ILK), a recently identified cytoplasmic serine/threonine protein kinase that binds to the cytoplasmic domain of beta(1) integrin, has an important role in transmembrane signal transduction via integrins. We report that ILK is expressed in N1E-115 cells, the expression levels of which are constant under both normal and differentiating conditions. A stable transfection of a kinase-deficient mutant of ILK (DN-ILK) results in inhibition of neurite outgrowth in serum-starved N1E-115 cells grown on laminin. On the other hand, a transient expression of wild type ILK stimulated neurite outgrowth. The ILK activity in the parental cells was transiently activated after seeding on the laminin matrix, whereas that in the DN-ILK-transfected cells was not. These results suggest that transient activation of ILK is required for neurite outgrowth in serum-starved N1E-115 cells on laminin. Under the same conditions, p38 mitogen-activated protein (MAP) kinase, but neither MAP kinase/extracellular signal-regulated kinase kinase (MEK) nor extracellular signal-regulated kinases (ERK), was transiently activated after N1E-115 cell attachment to laminin, but not in the DN-ILK-expressed cells. The time course of p38 MAP kinase activation was very similar to that of ILK activation. Furthermore, a p38 MAP kinase inhibitor, SB203580, significantly blocked neurite outgrowth. Thus, activation of p38 MAP kinase is involved in ILK-mediated signal transduction leading to integrin-dependent neurite outgrowth in N1E-115 cells.     

Changes in S-type lectin localization in neuroblastoma cells (N1E115) upon differentiation

The distribution of a 14.4 kDa S-type lectin was examined in murine neuroblastoma cells, either undifferentiated or after differentiation induced by dibutyryl-cyclic adenosine monophosphate. In undifferentiated cells the immunoreactivity was detected extracellularly, associated with the plasma membrane and in bulges released into the extracellular milieu. Important modifications of the lectin localization were associated with the differentiation process that induced an increased cytosolic expression and a decreased externalization. Possible functions for the lectin expressed intracellularly in the differentiated cells are also considered.     

Copine1 C2 domains have a critical calcium-independent role in the neuronal differentiation of hippocampal progenitor HiB5 cells

Copine1 (CPNE1) has tandem C2 domains and an A domain and is known as a calcium-dependent membrane-binding protein that regulates signal transduction and membrane trafficking. We previously demonstrated that CPNE1 directly induces neuronal differentiation via Akt phosphorylation in the hippocampal progenitor cell line, HiB5. To determine which region of CPNE1 is related to HiB5 cell neurite outgrowth, we constructed several mutants. Our results show that over-expression of each C2 domain of CPNE1 increased neurite outgrowth and expression of the neuronal marker protein neurofilament (NF). Even though protein localization of the calcium binding-deficient mutant of CPNE1 was not affected by ionomycin, this mutant increased neurite outgrowth and NF expression in HiB5 cells. Furthermore, Akt phosphorylation was increased by over-expression of the calcium binding-deficient CPNE1 mutant. These results suggest that neither cellular calcium levels nor the localization of CPNE1 affect its function in neuronal differentiation. Collectively, our findings indicating that the C2 domains of CPNE1 play a calcium-independent role in regulating the neuronal differentiation of HiB5 cells.     

Differentiation-dependent expression of hypothetical proteins in the neuroblastoma cell line N1E-115

Several protein cascades, including signaling, cytoskeletal, chaperones, metabolic, and antioxidant proteins, have been shown to be involved in the process of neuronal differentiation (ND) of neuroblastoma cell lines. No systematic approach to detect hitherto unknown and unnamed proteins or structures that have been predicted upon nucleic acid sequences in ND has been published so far. We therefore decided to screen hypothetical protein (HP) expression by protein profiling. Two-dimensional gel electrophoresis with subsequent matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF) identification was used for expression analysis of undifferentiated and dimethylsulfoxide-induced neuronally differentiated N1E-115 cells. We unambiguously identified six HPs: Q8C520, Q99LF4, Q9CXS1, Q9DAF8, Q91WT0, and Q8C5G2. A prefoldin domain in Q91WT0, a t-SNARE domain in Q9CXS1, and a bromodomain were observed in Q8C5G2. For the three remaining proteins, no putative function using Pfam, BLOCKS, PROSITE, PRINTS, InterPro, Superfamily, CoPS, and ExPASy could be assigned. While two proteins were present in both cell lines, Q9CXS1 was switched off (i.e., undetectably low) in differentiated cells only, and Q9DAF8, Q91WT0, and Q8C5G2 were switched on in differentiated cells exclusively. Herein, using a proteomic approach suitable for screening and identification of HP, we present HP structures that have been only predicted so far based upon nucleic acid sequences. The four differentially regulated HPs may play a putative role in the process of ND.     

SIRT1 regulates tyrosine hydroxylase expression and differentiation of neuroblastoma cells via FOXO3a

To examine the function of SIRT1 in neuronal differentiation, we employed all-trans retinoic acid (ATRA)-induced differentiation of neuroblastoma cells. Nicotinamide inhibited neurite outgrowth and tyrosine hydroxylase (TH) expression. Inhibition of PARP or histone deacetylase did not inhibit TH expression, showing the effect to be SIRT1 specific. Expression of FOXO3a and its target proteins were increased during the differentiation and reduced by nicotinamide. FOXO3a deacetylation was increased by ATRA and blocked by nicotinamide. SIRT1 and FOXO3a siRNA inhibited ATRA-induced up-regulation of TH and differentiation. Taken together, these results indicate that SIRT1 is involved in ATRA-induced differentiation of neuroblastoma cells via FOXO3a.     

Mitosis-dependent protein expression in neuroblastoma cell line N1E-115

Systematic work on differential protein expression in mitosis is limited, and we therefore used neuroblastoma cells (N1E-115) incubated with either colcemid or nocodazole to arrest mitosis. Proteins were identified by MALDI-TOF/TOF and nano-LC-ESI-MS/MS with subsequent quantification of spot volumes with specific software. Immunoblotting was used for verification of selected proteins. Levels of 10 individual proteins were increased and levels of 6 proteins were decreased concordantly by both treatments. These proteins were constituents of heat shock and chaperone, cytoskeleton, proteasomal, heterochromatin, and DNA replication signaling as well as housekeeping and metabolic systems. Identification of mitosis-dependent proteins is of importance for the interpretation of previous work and for designing future experiments.